Fatty acids are the main components of lipids [1]. Fatty acids can be categorized into two groups based on their chemical structures: saturated and unsaturated. Unsaturated fatty acids can be further divided into monounsaturated or polyunsaturated fatty acids. Another classification of fatty acids is based on whether they can be synthesized by the human body: nonessential fatty acids can be synthesized by the human body while essential fatty acids must be obtained from diet. Omega-3 (ω-3) and omega-6(ω-6) polyunsaturated fatty acids are two main types of essential fatty acids that can offer significant nutritional benefits to the human body [2]. Fish products are major sources of ω-3 fatty acids, and ω-6 fatty acids are mainly from grass-fed meat. 

The biosynthesis of polyunsaturated fatty acids is a complex process that involves two key processes: dehydrogenation reactions and fatty acid carbon chain elongation [4]. Desaturase and fatty acid carbon chain elongation enzymes work together to catalyze the two processes. Dehydrogenation is a reaction that uses the fat-1 gene, which can encode ω-3 desaturase, catalyzing the conversion of ω -6 polyunsaturated fatty acids (ω-6 PUFAs) into the equivalent omega-3 polyunsaturated fatty acids (ω -3 PUFAs) [5]. Elongation of long-chain polyunsaturated fatty acids like C22 can also be mediated by ELOVL5, a member of the elongase of very long-chain fatty acids (ELOVL) family [4].

 

ω-3 polyunsaturated fatty acids are known for their beneficial effects on enhancing cardiovascular and neurological health [3]. Therefore, health institutions recommend daily uptakes of ω-3 and ω-6 fatty with a ratio of . However, the diet habit of humans has changed drastically nowadays, with an increase in saturated fatty acids uptake and higher uptake of ω-6 fatty acids than ω-3 fatty acids, rresulting inain   ratio of 15:1. The imbalanced ratio between the two essential fatty acids may increase the risks of cardiovascular and neurological diseases as well as cancer and inflammatory disorders [2]. Moreover, overfishing has exceeded the sustainable capacity of natural fish stocks, leading to an inadequate uptake of ω-3 fatty acids from traditional sources [2].

Therefore, there is an increasing requirement for sustainable sources of ω-3 polyunsaturated acids. A potential solution is to increase the production of ω-3 polyunsaturated fatty acids through the terrestrial system. Some published literature indicates the feasibility of achieving this through genetic engineering using plasmid transformation [3]. DPA (docosapentaenoic acid) is often the third most prevalent ω-3 fatty acid found in fish oil. Some studies have found DPA plays a role in imparting health benefits [1].

 

Our project aims to design and construct fat-1 and ELOVL5 prokaryotic expression plasmids, as well as express ω-3 desaturase and ELOVL5 in the E. coli cells to synthesize DPA. To achieve this, recombined plasmids, namely pET-28a-fat1, pET-28a-ELOVL5, pET-28a-fat1-ELOVL5, and pET-28a-GFP-fat1-ELOVL5, will be constructed, transformed and expressed into DH5α and BL21 competent cells. If plasmid construction is successful, the presence of these two enzymes could enable the production of ω-3 polyunsaturated fatty acids in E. coli. The success of this initial experiment on E. coli suggest the potential application in oil-producing plants, allowing their seeds to be enriched with omega-3 fatty acids and can be consumed by human, alleviating the shortage of marine sources for ω-3 fatty acids.

 

[1] De Carvalho, C. C., & Caramujo, M. J. (2018). The various roles of fatty acids. Molecules, 23(10), 2583.

[2] Amjad Khan, W., Chun-Mei, H., Khan, N., Iqbal, A., Lyu, S. W., & Shah, F. (2017). Bioengineered plants can be a useful source of omega-3 fatty acids. BioMed research international, 2017.

[3] Colombo, S. M., Campbell, L. G., Murphy, E. J., Martin, S. L., & Arts, M. T. (2018). Potential for novel production of omega-3 long-chain fatty acids by genetically engineered oilseed plants to alter terrestrial ecosystem dynamics. Agricultural Systems, 164, 31-37.

[4] Uttaro, A. D. (2006). Biosynthesis of polyunsaturated fatty acids in lower eukaryotes. IUBMB life, 58(10), 563-571.

[5] Ji, S., Hardy, R. W., & Wood, P. A. (2009). Transgenic expression of n‐3 fatty acid desaturase (fat‐1) in C57/BL6 mice: Effects on glucose homeostasis and body weight. Journal of Cellular Biochemistry, 107(4), 809-817.